Glutamic acid is a carrier for hydrazine during the biosyntheses of fosfazinomycin and kinamycin

Abstract

Fosfazinomycin and kinamycin are natural products that contain nitrogennitrogen (N-N) bonds but that are otherwise structurally unrelated. Despite their considerable structural differences, their biosynthetic gene clusters share a set of genes predicted to facilitate N-N bond formation. In this study, we show that for both compounds, one of the nitrogen atoms in the N-N bond originates from nitrous acid. Furthermore, we show that for both compounds, an acetylhydrazine biosynthetic synthon is generated first and then funneled via a glutamyl carrier into the respective biosynthetic pathways. Therefore, unlike other pathways to N-N bond-containing natural products wherein the N-N bond is formed directly on a biosynthetic intermediate, during the biosyntheses of fosfazinomycin, kinamycin, and related compounds, the N-N bond is made in an independent pathway that forms a branch of a convergent route to structurally complex natural products.More than 200 natural products containing nitrogen-nitrogen (N-N) bonds have been identified with various bioactivities (Fig. 1a) 1 . For instance, streptozotocin, a nitrosamine-containing compound, exhibits cytotoxic activity and is currently deployed clinically 2 , and valanimycin, featuring an azoxy group, displays both antimicrobial and cytotoxic activities 1 . Other examples are azamerone containing a pyridazine, kutzneride with a piperazic acid 2, 3 , the fosfazinomycins having a phosphonohydrazide 4 , and a group of molecules including cremeomycin, the lomaiviticins and the kinamycins containing diazo groups 1 .

Nitric oxide as a source for bacterial triazole biosynthesis

“…We fed strain Streptomyces albus subsp. pathocidicus ATCC 14510 with the fully 15 Supplementary Fig. 1), establishing that compound 1 retains all the five nitrogen atoms (N-1, N-2, N-3, N-7, N-9) from a single molecule of 15 N 5 -GTP.…”

“…The result revealed a new mass signal (m/z 158, [M + H] + ) for 1, with a +5 Da mass shift compared with unlabeled 1 (m/z 153, [M + H] + ) (Supplementary Fig. 1), establishing that compound 1 retains all the five nitrogen atoms (N-1, N-2, N-3, N-7, N-9) from a single molecule of 15 N 5 -GTP. Together with the earlier isotope feeding experiments, these results strongly indicate that construction of 1 involves the removal of the C( 8)-H group of guanine, which could occur at the level of free nucleobase or in its nucleoside or nucleotide forms, and followed by re-cyclization with a nitrogen atom from an unknown source.…”

“…We fed strain Streptomyces albus subsp. pathocidicus ATCC 14510 with the fully 15 N-labeled guanosine triphosphate ( 15 N 5 -GTP) at a final concentration of 1 mM, and analyzed the resulting 1 by LC-MS. The result revealed a new mass signal (m/z 158, [M + H] + ) for 1, with a +5 Da mass shift compared with unlabeled 1 (m/z 153, [M + H] + ) (Supplementary Fig.…”

“…The most frequently used precursors for N-N bond formation are hydroxylamines (such as L-N 5 -OH-Ornthine in piperazate pathway 9 , N-isobutylhydrdoxylamine in valanimycin pathway 37 , and L-N 6 -OH-lysine in the pathways of s56-p1 14 , pyrazomycin 16 and formycin 17,38 ) and nitric acid, which derives from the α-amine of L-aspartic acid by the enzyme pair CreE and CreD 10 . The flavin monooxygenase CreE and nitrosuccinate lyase CreD were first characterized in the cremeomycin pathway, and their homologs were subsequently identified in the pathways of fosfazinomycin, and kinamycin, triacsins and alanosine 15,[21][22][23] .…”

“…While the biosynthetic routes to natural products containing a N-N bond have received great interest and are starting to be revealed, how a triazole ring is assembled in nature remains unknown [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] . Elucidation of its biosynthetic mechanism could likely be helpful to the design of synthetic biology approaches for producing triazoles.…”